Structural and Functional Insights into the Regulation of Helicobacter pylori CPA Activity by an Evolutionarily Conserved Motif.

Abstract

N-Carbamoyl putrescine amidase (CPA) is a key enzyme in polyamine synthesis and plays an essential role in cell growth and proliferation in plants and many bacteria. While CPA is absent in humans, its presence in Helicobacter pylori makes it a potential therapeutic target. Despite its biological importance, CPA's structural and functional characteristics are not fully understood. The (-¹⁵²CWDQW¹⁵⁶-) motif in H. pylori CPA is conserved across homologues, where tryptophan residues are critical for oligomerization. This motif forms a short helix near the active site, suggesting a functional role for helix formation. Using computational and solution-based experiments, we show the significance of short-helix formation in CPA function. Unlike the wild-type and the Cys152Ala mutant, which form octamers, the Cys152Pro variant disrupts the helix formation and produces dimers. This indicates that short-helix construction is crucial for producing octamers. Furthermore, we observed that residues Asp154 and Tyr157 interact with His117 within the same monomer. Notably, single Ala mutations of these residues resulted in dimers with significantly reduced catalytic activity despite maintaining helix formation. This finding underscores the importance of the interaction network (Asp154-His117-Tyr157) in stabilizing the helix's orientation and promoting octamerization. We also observed that octamerization enhances protein stability, as evidenced by a 10° increase in T_m. Targeting the short helix and its surrounding region, we identified a small-molecule CPA inhibitor that effectively reduced enzymatic activity. These findings reveal a new regulatory mechanism where helix formation and oligomerization are crucial to CPA function, providing a foundation for developing more potent therapeutic inhibitors against H. pylori.